Expression of proteins and carbohydrates capable of promoting bacterial growth in multicellular communities called biofilms is an important virulence property of a number of human pathogens. In Pseudomonas aeruginosa and in many other gram-negative bacteria, the formation of these surface structures is controlled by the cellular levels of the second messenger cyclic di GMP (c-di-GMP). The concentrations of c-di-GMP are determined by the antagonistic activities of two classes of enzymes. Diguanylate cyclases (DGCs) catalyze the formation of c-di-GMP while phosphodiesterases (PDEs) are responsible for the degradation of this regulatory dinucleotide. In this project a chemical approach will be used to identify and characterize inhibitors of DGCs and PDEs. The P. aeruginosa WspR regulates the production of several biofilm components including the glucan-rich PEL polysaccharide, while RocR, a PDE, has been shown to regulate the production of the CupC fimbrial adhesin. Recombinant forms of these two highly active proteins were purified in large quantities and were used to develop specific enzymatic assay for c-di-GMP synthesis and degradation. In Aim 1 of this proposal, we will utilize synthetic analogues of c-di-GMP prepared by organic synthesis or by enzymatic synthesis using hyperactive mutants of WspR. They will be tested not only as inhibitors of WspR and RocR but also for their activities against other DGCs and PDEs produced by P. aeruginosa, with the objective of identifying broad-spectrum inhibitors. We will also test the candidate inhibitors for their ability to bind c-di-GMP receptors and interfere with the binding of the natural c-di-GMP ligands. Structural derivatives of active compounds will be ordered and tested to establish structure-activity relationships of various chemical moieties. In Aim 2 we will characterize the active compounds for in vivo inhibition of the target enzymes in P. aeruginosa and assess their biological activities on biofilm development. The compounds will be also tested as potential substrates for elimination by specific efflux pumps produced by this organism. In addition to providing valuable research tools to probe the role of the c-di-GMP in virulence, the identification of DGC and PDE inhibitors should provide the basis for a preclinical program directed towards the development of these reagents into broad-spectrum antimicrobial agents targeting biofilm formation.